Arginase
Arginase is a manganese metalloenzyme containing a metal-activated hydroxide ion, a critical nucleophile in metalloenzymes that catalyze hydrolysis or hydration reactions. Arginase converts naturally occurring arginine into ornithine and urea. The enzyme exits in many living organisms, including bacteria and humans (Jenkinson et al., 1996, Comp Biochem Physiol B Biochem Mol Biol, 114:107-32).
Pegylation of Arginase
Arginase may be used as a therapeutic agent and administered parenterally for various indications. However, parenterally administrated arginase, which is a protein, may be immunogenic and typically has a short pharmacological half-life. Consequently, it can be difficult to achieve therapeutically useful blood levels of the proteins in patients. These problems may be overcome by conjugating the proteins to polymers such as polyethylene glycol (PEG).
Covalent attachment of the inert, non-toxic, biodegradable polymer PEG, to molecules has important applications in biotechnology and medicine. Pegylation of biologically and pharmaceutically active proteins has been reported to improve pharmacokinetics, resulting in sustained duration, improve safety (e.g. lower toxicity, immunogenicity and antigenicity), increase efficacy, decrease dosing frequency, improve drug solubility and stability, reduce proteolysis, and facilitate controlled drug release (Roberts et al., 2002, Adv Drug Deliv Rev, 54:459-76; Harris & Chess, 2003, Nat Rev Drug Discov, 2:214-221).
PEG-protein conjugates produced by conventional methods in the art contain heterogeneous species, each being attached with a variable number of PEG molecules, ranging from zero to the number of amino groups that the protein has. Even for species that has the same number of PEG molecule attached, the site of attachment on the protein may vary from species to species. Such non-specific pegylation, however, can result in conjugates that are partially or virtually inactive. Reduction of activity may be caused by shielding the protein's active receptor binding domain when the PEG is attached at an improper site. Thus, there is a clear need for a better way of producing homogeneously pegylated protein molecules which retain the activity of the parent protein and making possible the administration of correct and consistent dosages necessary for clinical uses.
Cancer Treatment Via Amino Acid Deprivation
Amino acid deprivation therapy is an effective means for the treatment of some cancers. Although normal cells do not require arginine, many cancer cell lines are auxotrophic for this amino acid. Many lines of evidence have shown that in vitro arginine depletion, either with an arginine-degrading enzyme or by using an arginine-deficient medium, leads to rapid destruction of a wide range of cancer cells (Scott et al., 2000, Br J Cancer, 83:800-10). But direct use of enzymes, which are proteins, has problems of immunogenicity, antigenicity and short circulating half-life.
Inhibition of Virus by Arginine Deprivation
Viral infections are among the leading causes of death with millions of deaths each year being directly attributable to several viruses including hepatitis and human immunodeficiency virus (HIV). However, there are several problems with current anti-viral therapies. First, there are relatively few effective antiviral drugs. Many of the existing anti-viral agents cause adverse or undesirable side-effects. Most effective therapies (such as vaccination) are highly specific for only a single strain of virus. Frequently the virus undergoes mutation such that it becomes resistant to either the drug or vaccine. There is a need for methods for inhibiting viral replication which do not have the problems associated with the prior art.
Many studies over the last 30 years have demonstrated that extracellular arginine is required for viral replication in vitro. Historically this has been accomplished by making tissue culture media deficient in arginine and dialyzing the serum used as a supplement in order to achieve arginine free medium. Using this methodology to achieve arginine deprivation results in inhibition of replication of a large number of diverse families of viruses including: adenovirus (Rouse et al., 1963, Virology, 20:357-365), herpes virus (Tankersley, 1964, J Bacteriol, 87: 609-13).
Human Immunodeficiency Virus (HIV)
Acquired immune deficiency syndrome (AIDS) is a fatal disease, reported cases of which have increased dramatically within the past several years. The AIDS virus was first identified in 1983. It has been known by several names and acronyms. It is the third known T-lymphotropic virus (HTLV-III), and it has the capacity to replicate within cells of the immune system, causing profound cell destruction. The AIDS virus is a retrovirus, a virus that uses reverse transcriptase during replication. Two distinct families of HIV have been described to date, namely HIV-1 and HIV-2. The acronym “HIV” is used herein to refer to human immunodeficiency viruses generically. HIV replication is believed to be arginine-dependent, depletion of which would thus inhibit HIV replication.